Speaker
Description
Background and Aims
Radiotherapy (RT) is one of the most important approaches to treat cancer, but normal tissue tolerance limits the delivery of curative doses to the tumour. By using dose distributions that differ from the flat profiles employed in conventional RT, spatially fractionated radiotherapy (SFRT) techniques have demonstrated a remarkable capacity to spare normal tissue while preserving tumour control. Despite growing experimental evidence, the molecular mechanisms underlying these differential biological responses remain unclear. In this context, synchrotron radiation-based Fourier Transform Infrared microspectroscopy (SR-FTIRM) provides label-free biochemical characterization of irradiated cells. This work, based on multiple experiments, aimed to characterize the biomolecular alterations induced in healthy and tumour cells after being irradiated with different SFRT configurations.
Methods
Cells were irradiated with conventional RT and SFRT under multiple irradiation configurations, including beam type, dose, post-irradiation time points, and irradiation geometries. SR-FTIRM measurements were performed at the MIRAS beamline of the ALBA Synchrotron, in transmission mode using a 10 × 10 µm² aperture. The high brilliance of synchrotron radiation allowed high signal-to-noise spectral imaging at subcellular resolution. Spectra were collected in the 900-3800 cm-1 range (mid-infrared), covering proteins, lipids, carbohydrates, and nucleic acids. Spectral datasets were analysed using multivariate statistical approaches as principal component analysis (PCA), selected band spectral ratios, and supervised machine learning classification, including PCA-LDA (PCA-linear discriminant analysis) and PLS-DA (Partial Least Squares-discriminant analysis).
Results
Distinct modality-specific biochemical fingerprints were identified across irradiation conditions and cell lines. In healthy cells, conventional RT generally showed greater spectral differences relative to non-irradiated cells. The greatest modifications due to SFRT were observed for tumour cells, in the fingerprint region (950-1800 cm-1), involving alterations in the secondary structure of proteins (amide I and II bands, 1500-1700 cm-1) and nucleic acids (950-1400 cm-1). The carbonyl group (near 1740 cm-1) suggested modality-dependent alterations, possibly associated with changes in the degree of oxidative stress, membrane integrity and cell death. Classification models achieved high accuracy, precision, and recall (0.82 – 1.00) for the fingerprint region, supporting the presence of relevant irradiation-induced alterations. Additional differences between SFRT and conventional RT were also observed in the lipids region (2800-3000 cm-1), involving symmetric and asymmetric CH2 and CH3 bands, potentially reflecting changes in the lipid chain length and cell membrane due to oxidative stress or cell death mechanisms.
Conclusions
SR-FTIRM provides a powerful bioanalytical tool for resolving the biochemical complexity of SFRT. Its potential is reinforced by the high performance in spectral classification employing machine learning algorithms.